A small-molecule P2RX7 activator promotes anti-tumor immune responses and sensitizes lung tumor to immunotherapy

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A small-molecule P2RX7 activator promotes anti-tumor

immune responses and sensitizes lung tumor to

immunotherapy

Laetitia Douguet, Serena Janho Dit Hreich, Jonathan Benzaquen, Laetitia

Seguin, Thierry Juhel, Xavier Dezitter, Christophe Duranton, Bernhard

Ryffel, Jean Kanellopoulos, Cecile Delarasse, et al.

To cite this version:

Laetitia Douguet, Serena Janho Dit Hreich, Jonathan Benzaquen, Laetitia Seguin, Thierry Juhel, et

al.. A small-molecule P2RX7 activator promotes anti-tumor immune responses and sensitizes lung

tumor to immunotherapy. Nature Communications, Nature Publishing Group, 2021, 12 (1), pp.653.

�10.1038/s41467-021-20912-2�. �hal-03126997�

A small-molecule P2RX7 activator promotes

anti-tumor immune responses and sensitizes lung

tumor to immunotherapy

Laetitia Douguet

1,15

✉

, Serena Janho dit Hreich

1,2,3,15

, Jonathan Benzaquen

1,2,3

, Laetitia Seguin

1,2

,

Thierry Juhel

1

, Xavier Dezitter

4,5

, Christophe Duranton

6

, Bernhard Ryffel

7

, Jean Kanellopoulos

8

,

Cecile Delarasse

9

, Nicolas Renault

4,5

, Christophe Furman

4,5

, Germain Homerin

4,10

, Chloé Féral

1,2

,

Julien Cher

fils-Vicini

1

, Régis Millet

4,5

, Sahil Adriouch

11

, Alina Ghinet

4,10,12

, Paul Hofman

1,2,13,14

&

Valérie Vouret-Craviari

1,2,3

✉

Only a subpopulation of non-small cell lung cancer (NSCLC) patients responds to

immu-notherapies, highlighting the urgent need to develop therapeutic strategies to improve patient

outcome. We develop a chemical positive modulator (HEI3090) of the purinergic P2RX7

receptor that potentiates

αPD-1 treatment to effectively control the growth of lung tumors in

transplantable and oncogene-induced mouse models and triggers long lasting antitumor

immune responses. Mechanistically, the molecule stimulates dendritic P2RX7-expressing

cells to generate IL-18 which leads to the production of IFN-

γ by Natural Killer and CD4

+

T cells within tumors. Combined with immune checkpoint inhibitor, the molecule induces a

complete tumor regression in 80% of LLC tumor-bearing mice. Cured mice are also protected

against tumor re-challenge due to a CD8-dependent protective response. Hence,

combina-tion treatment of small-molecule P2RX7 activator followed by immune checkpoint inhibitor

represents a strategy that may be active against NSCLC.

https://doi.org/10.1038/s41467-021-20912-2

OPEN

1_{Université Côte d’Azur, CNRS, INSERM, IRCAN, Nice, France.}2_{FHU OncoAge, Nice, France.}3_{Centre Antoine Lacassagne, Nice, France.}4_{Inserm, CHU Lille,}

U1286—Infinite—Institute for Translational Research in Inflammation, University of Lille, Lille, France.5Institut de Chimie Pharmaceutique Albert Lespagnol, IFR114, Lille, France.6Université Côte d’Azur, CNRS, INSERM, LP2M, Nice, France.7INEM—UMR7355, Institute of Molecular Immunology and

Neurogenetic, University and CNRS, Orleans, France.8Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette Cedex, France.9INSERM, CNRS, Institut de la Vision, Sorbonne Université, Paris, France.10Hautes Etudes d’Ingénieur (HEI), JUNIA, UC Lille, Laboratoire de Chimie Durable et Santé, Lille, France.11_{Institute for Research and Innovation in Biomedicine, Normandie University, Rouen, France.}12_{Faculty of Chemistry,}

‘Al. I. Cuza’ University of Iasi, Iasi, Romania.13_{Hospital-Related Biobank (BB-0033-00025), Pasteur Hospital, Nice, France.}14_{Laboratory of Clinical and}

Experimental Pathology and Biobank, Pasteur Hospital, Nice, France.15_{These authors contributed equally: Laetitia Douguet, Serena Janho dit Hreich.}

✉email:ldouguet@gmail.com;valerie.vouret@univ-cotedazur.fr

123456789

D

espite new biological insights and recent therapeutic

advances, many tumors remain resistant to treatments,

leading to premature death of the patient. This is

parti-cularly true for lung cancer, which is the leading cause of cancer

death for men and women worldwide. The 5-year survival rate for

patients with any type of lung cancer is around 20%, which

dramatically drops to 6% for metastatic lung cancers. Recent

advances in effective therapies such as targeted therapies and

immunotherapies have revolutionized lung cancer treatments

1

_.

However, it is limited to a small percentage of patients and

alternative approaches are urgently needed to improve patient

outcome.

The P2RX7 receptor (also called P2X7R) is an ATP-gated ion

channel composed of three protein subunits (encoded by the

P2RX7 gene), which is expressed predominantly in immune cells

and in some tumor cells

2

_{. Activation of P2RX7 by high doses of}

extracellular ATP (eATP) leads to Na

+

and Ca

2+

influx, and,

after prolonged activation, to the opening of a larger

con-ductance membrane pore. One consequence of this large pore

opening, a unique characteristic of P2RX7, is to induce cell

death in eATP rich microenvironments. Noteworthy, such high

doses of eATP are present in the inflammatory and tumor

microenvironments (TMEs)

3

_{. P2RX7 functions are largely}

described in immune cells, where it is involved in NLRP3

acti-vation to induce the maturation and secretion of 1β and

IL-18 pro-inflammatory cytokines by macrophages and dendritic

cells (DCs)

4

. In line, several P2RX7 inhibitors have been

developed with the aim to treat inflammatory diseases. In

addition to its ability to

finely tune the amplitude of the

inflammatory response

5

_{, P2RX7 has been shown to orchestrate}

immunogenic cell death (ICD) and to potentiate DC activation

and ability to present tumor antigens to T cells

6

_{. Among}

immune cells, regulatory T cells (Treg) are highly sensitive to

P2RX7-induced cell death and, in the presence of eATP, P2RX7

negatively regulates their number and their suppressive

func-tion

7

_{. Such response can participate in P2RX7-dependent}

immune surveillance by unleashing the effector functions of

adaptive immune T cells

8

_{. Therefore, P2RX7 has been proposed}

to represent a positive modulator of antitumor immune

response. This is in agreement with data from our group

showing that P2RX7-deficient mice are more sensitive to

colitis-associated cancer

9

_{. Also, in this model, we noticed that}

trans-planted Lewis lung carcinoma (LLC) tumors grew faster in line

with the

findings of Adinolfi et al. using transplanted B16

melanoma and CT26 colon carcinoma tumors

10

_{. Collectively,}

these results support the notion that P2RX7 expression by host

immune cells coordinates antitumor immune response.

Capture of tumor antigens by antigen-presenting DC is a key

step in immune surveillance. Activated DCs present tumor

antigens to naïve T cells leading to their activation and

differ-entiation in effector T cells. Tumor infiltrated effector T cells and

NK cells can recognize and kill tumor cells resulting in the release

of additional tumor antigens and amplification of the immune

response. However, this response is often inhibited by

immuno-suppressive mechanisms present within the TME. Different

mechanisms sustain tumor escape as the reduced immune

recognition of tumors due to the absence of tumor antigens, or

the loss of MHC-I and related molecules, the increased resistance

of tumor cells edited by the immune responses, and the

devel-opment of a favorable TME associated with the presence of

immunosuppressive cytokines and growth factors (such as

VEGF, TGF-β) or the expression of checkpoint inhibitors such as

PD-1/PD-L1

11

. Inhibitory checkpoint inhibitors (αPD-1/PD-L1

and anti-CTLA-4) are used in daily practice for the treatment

of advanced malignancies, including melanoma and

non-small cell lung cancer (NSCLC)

12

. These antibodies reduce

immunosuppression and reactivate cytotoxic effector cell

func-tions to elicit robust antitumor responses

13,14

. High response rate

to

αPD-1/PD-L1 therapy is often associated with immune

inflamed cancer phenotype characterized by the presence in the

TME of both CD4

+

and CD8

+

T cells, PD-L1 expression on

infiltrating immune and tumor cells and many pro-inflammatory

and effector cytokines, such as IFN-γ

15

_{. Noteworthy, only few}

cancer patients achieve a response with anti-immune checkpoint

administered as single-agent

16

_{, suggesting that strategies based on}

combined therapies would likely enhance antitumor efficacy and

immunity.

Despite the role of P2RX7 in stimulating antitumor immunity

and the observation that tumor development is more aggressive

in p2rx7-deficient animals

9

_{, it is currently not known whether}

P2RX7 activation can modulate tumor progression in vivo. The

purpose of this study is to investigate the effect of a positive

modulator (PM) of P2RX7 on lung tumor fate. To do so, we use

syngeneic immunocompetent tumor mice models and show that

activation of P2RX7 improves mice survival. Mechanistically,

activation of P2RX7 leads to increased production of IL-18 in a

NLRP3-dependent manner, which in turn activates NK and

CD4

+

T cells to produce IFN-γ and consequently increases tumor

immunogenicity. Finally, activation of P2RX7 combined with

αPD-1 immune checkpoint inhibitor allows tumor regression,

followed by the establishment of a robust immunological memory

response.

Results

HEI3090 is a positive modulator of P2RX7. In order to identify

positive modulator of P2RX7, 120 compounds from the HEI’s

proprietary chemical library were screened for their ability to

increase P2RX7-mediated intracellular calcium concentration

during external ATP exposure. We

first produced an HEK cell

line expressing the cDNA encoding for P2RX7 from C57BL/6

origin (HEK mP2RX7) and determined the minimal dose of ATP

(333 µM) that should be used to initiate an increase in Ca

2+

concentration. We tested

five promising compounds and

identi-fied HEI3090 as a hit (patent WO2019185868A1). HEI3090

corresponds to a pyrrolidin-2-one derivative decorated with a

6-chloropyridin-3-yl-amide in position 1 and with a

2,4-dichlor-obenzylamide moiety in position 5 (Fig.

1

a and Supplementary

Fig. 1). HEI3090 alone showed no toxic activity, was unable to

induce intracellular Ca

2+

variation, and required the presence of

eATP to rapidly and dose dependently enhance the

P2RX7-mediated intracellular calcium concentration (Fig.

1

b, c). The

maximum effect of HEI3090 was observed at 250 nM, which is in

the range of doses identified in pharmacokinetic analysis

(Fig.

1

g). HEI3090 action required the expression of P2RX7, since

HEK cells transfected with empty plasmid (pcDNA6) showed no

increase in intracellular calcium concentration (Fig.

1

c). P2RX7

has the unique capacity to form a large pore under eATP

sti-mulation. Large pore opening of P2RX7 was assayed with the

quantification of the uptake of the fluorescent TO-PRO-3 dye. As

expected, HEI3090 alone had no effect and required eATP

sti-mulation to enhance TO-PRO-3 entry within the cells. HEI3090

increased by 2.5-fold the large pore opening (Fig.

1

d). The rapid

uptake of TO-PRO-3 was consistent with direct P2RX7 activation

rather than ATP/P2RX7-induced cell death (Fig.

1

e). We also

tested HEI3090’s effect on splenocytes expressing physiologic

levels of P2RX7 (Fig.

1

f). In these immune cells, HEI3090 alone

did not affect Fluo-4-AM nor TO-PRO-3 uptake. However, in the

presence of eATP, HEI3090 enhanced Ca

2+

influx and

TO-PRO-3 uptake. We also showed that HEITO-PRO-3090 required the expression

of P2RX7, since its effect was lost in splenocytes isolated from

p2rx7

−/−

mice.

Collectively these results demonstrate that HEI3090 requires

P2RX7 expression to be active and enhances eATP-induced

P2RX7 activation.

HEI3090 inhibits tumor growth and enhances antitumor

effi-cacy of

αPD-1 treatment. We previously suggested that P2RX7

expression might favor the activation of immune responses

9

_{. We}

therefore evaluated the immuno-stimulatory effect and antitumor

efficacy of HEI3090 in vivo, hypothesizing that the high level of

eATP contained within the TME

17

_{would be sufficient to stimulate}

P2RX7. To do so, we used syngeneic LLC and B16-F10 melanoma

cell lines expressing P2RX7 (Supplementary Fig. 2). Vehicle or

HEI3090 (1.5 mg/kg) was administered concomitantly to LLC

tumor cell injection and mice were treated daily for 11 days. Mice

treated with HEI3090 displayed significantly reduced tumor

growth and more than fourfold decrease in tumor weight (Fig.

2

a

and Supplementary Fig. 3). We next tested the efficacy of HEI3090

to inhibit tumor growth in a therapeutic model, in which

treat-ment started when tumor reached 10–15 mm

2

_{in size. HEI3090}

(3 mg/kg) inhibited tumor growth and increased by twofold the

median survival (Fig.

2

b). We also tested the effect of HEI3090 in

the melanoma B16-F10 tumor mouse model and observed the

same efficacy (Supplementary Fig. 4a, b).

Given the efficacy of HEI3090 to inhibit tumor growth, we next

evaluated the combination of HEI3090 and

αPD-1 antibody.

Fig. 1 HEI3090 enhances ATP-induced receptor channel activity. a Representation of HEI3090’s synthesis steps. b Modulation of ATP-induced intracellular Ca2+variation (F1/F0) in HEK293T-mP2RX7 cells (C57Bl/6 origin). After ten baseline cycles, ATP (333_{µm) and HEI3090 (250 nM) were} injected. Error bars are mean ± SEM (n = 3 independent experiments, 6 replicates). c Average Fluo-4-AM fluorescence intensities in HEK293T mP2RX7 or control HEK pcDNA6 measured 315 s after stimulation with ATP (333µM) and HEI3090 at concentrations of 25, 250, and 2.5 µM, as indicated in the color code. Data are presented as scatter dot plots ± SEM (n = 3 independent experiments and 6 replicates, two-tailed Mann–Whitney test). d Modulation of ATP-induced TO-PRO-3 uptake in HEK293T mP2RX7 cells (F1/F0) in cells treated with ATP and HEI3090 (25 nM). Error bars are mean ± SEM (n = 3 independent experiments, 4 replicates, two-tailed Mann–Whitney test). e Average fluorescence intensities in HEK293T mP2RX7 or control HEK pcDNA6 measured 10 min after stimulation with ATP and HEI3090 at concentrations of 25, 250, and 2.5µM, as indicated in the color code. Data are presented as scatter dot plots ± SEM (_{n = 3 independent experiments and 4 replicates, two-tailed Mann–Whitney test). f Left: average Fluo-4-AM fluorescence} intensities in WT orp2rx7−/−splenocytes stimulated with 50µM ATP measured at the plateau i.e., 10 min after stimulation. Data are presented as scatter dot plots ± SEM (n = 2 independent experiments and 4 replicates). Right: graph represents the percentage of TO-PRO-3 positive cells in splenocytes isolated from naïve WT or_p2rx7−/−mice. Data are presented as scatter dot plots ± SEM (_{n = 3 independent experiments in duplicate, two-tailed} Man–Whitney test). g Pharmacokinetic analysis of HEI3090 intraperitoneally injected in WT mice. Error bars are means ± SEM (n = 3 independent experiments in duplicate). Bars are mean ± SEM.p values: *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.

After tumor inoculations, mice were treated daily with HEI3090

or vehicle and

αPD-1 was administered at days 4, 7, 10, 13, and

16. While only 1 mouse out of the 16 mice treated with the

αPD-1

alone showed a tumor regression, 13 out of the 16 mice treated

with HEI3090

+ αPD-1 were tumor-free, suggesting that this

molecule increased the efficacy of immune checkpoint inhibitor

to induce effective antitumor immune responses and tumor

regression (Fig.

2

c). Importantly, only the combo treatment

allows a long-lasting improved survival of at least 340 days

(Fig.

2

c, right panel). The combo treatment also increased the

survival of mice grafted with B16-F10 tumors (Supplementary

Fig. 4c). As illustrated in Fig.

2

d, we tested the combo treatment

on the KRAS-driven lung cancer (LSL Kras

G12D

_{) model, which}

leads to adenocarcinomas 4 months after instillation of

adenoviruses expressing the Cre recombinase

18

_{. Whereas}

_αPD-1

treatment tends to reduce the number of ADC (Fig.

2

d), HEI3090

was able to enhance

αPD-1’s effects in this mouse model. Indeed,

tumor burden in mice treated with the combo treatment is

reduced by 60% compared to mice treated with

αPD-1 alone.

Accordingly, the cell number per mm

2

_{and the number of cells}

positively stained for the proliferation marker Ki67 were

decreased by 50% in lesion areas (Supplementary Fig. 4d). One

mouse out of the six treated with HEI3090 and

αPD-1 was

protected against adenocarcinoma formation.

DCs mediate the antitumor effect of HEI3090. LLC tumor cells

express an active P2RX7, since the presence of high doses of

eATP leads to an increase in intracellular Ca

2+

concentration,

which is blocked by the GSK1370319A P2RX7 inhibitor

19

(Sup-plementary Fig. 2a). To functionally investigate which cells are

targeted by HEI3090, we inoculated LLC in p2rx7

−/−

mice and

treated them with HEI3090. Whereas HEI3090 efficiently blocked

LLC tumor growth in WT mice (Fig.

2

a), the same treatment was

inefficient in p2rx7

−/−

_{mice, as tumor growth was}

indis-tinguishable in treated or untreated groups (Fig.

3

a). This result

suggests that HEI3090 requires P2RX7 expression by mouse host

cells to inhibit tumor growth. The importance of immune cells

was further confirmed by the demonstration that the antitumor

efficacy of HEI3090 was restored after adoptive transfer of WT

splenocytes into p2rx7

−/−

mice. DCs express P2RX7 and

orchestrate antitumor immunity. Purified DC from WT spleens

transferred into p2rx7

−/−

mice were able to restore the antitumor

effect of HEI3090 (Fig.

3

b). This experiment was further

sup-ported by the fact that phagocytic cells (macrophages and DC)

were required for HEI3090’s antitumor effect (Supplementary

Fig. 5a) and that macrophages are less implicated in HEI3090’s

effect in vivo since HEI3090 is still able to inhibit tumor growth

in p2rx7

fl/fl

LysM mice (Supplementary Fig. 5b).

Flow cytometry analyses revealed that the TME of mice treated

with HEI3090 were more infiltrated by immune cells than control

mice (Fig.

3

d). An increased infiltration of CD8

+

T cells was also

observed in the LSL-Kras

G12D

_{lung tumor mouse model (Fig.}

₃

_e).

Furthermore, we showed that HEI3090-treated mice showed

higher levels of P2RX7 on DC (Supplementary Fig. 5c). Deeper

characterization of immune cell infiltrate in the LLC tumor model

revealed that anti-CD3 staining of tumors from HEI3090-treated

mice contained four times more CD3

+

T cells than tumors from

vehicle-treated mice (Fig.

3

f). Whereas the proportion of

CD4

+

FOXP3

+

Treg cells was comparable between treated or

untreated mice (Fig.

3

g), we found fewer myeloid derived

suppressor cells (PMN-MDSCs) after HEI3090 therapy (Fig.

3

h)

and higher NK/PMN-MDSC and CD4/PMN-MDSC ratios

(Fig.

3

i) but the treatment failed to consistently increase the

CD8/PMN-MDSC ratio. We also showed that HEI3090 targets

immune cells in the low immunogenic B16-F10 melanoma

syngeneic mouse model, where it was able to increase antitumor

effector cells and decrease M-MDSCs infiltration (Supplementary

Fig. 6).

P2RX7 expressed by DC has been shown to link innate and

adaptive immune responses against dying tumor cells upon

chemotherapy-induced ICD and facilitate tumor antigens

pre-sentation to T cells

6

. We evaluated the capacity of HEI3090

treatment to kill tumor cells and concomitant stimulation of DC

maturation. Our results showed that HEI3090 is not an ICD

inducer (Supplementary Fig. 7).

The two tumor cell lines used in this study express different

levels of P2RX7 (Supplementary Fig. 2), yet HEI3090 required

P2RX7’s expressing immune cells to inhibit tumor growth in both

tumor mouse models. These results demonstrate that HEI3090

controls tumor growth by recruiting and activating

P2RX7-expressing immune cells, especially DC, within the TME to

initiate an effective antitumor immune response.

IL-18 is produced in response to HEI3090 treatment and is

required to mediate its antitumor activity. We then investigated

how the activation of P2RX7 enhanced antitumor immune

responses. In addition to increasing intracellular Ca

2+

con-centration and stimulating the formation of a large membrane

pore (see Fig.

1

), P2RX7’s activation is also known to activate the

NLRP3 inflammasome that leads to the activation of caspase-1

and consequently to the maturation and release of the

pro-inflammatory cytokines IL-1β and IL-18. We showed that

HEI3090 enhanced caspase-1 cleavage (Supplementary Fig. 8).

Whereas neutralization of IL-1β did not impact HEI3090’s

anti-tumor activity, neutralization of IL-18 suppressed the antianti-tumor

effect of HEI3090 (Fig.

4

a). This result was confirmed using

il18

−/−

mice in which HEI3090 had no impact on tumor growth

(Fig.

4

b). IHC staining of LLC tumors from HEI3090-treated

mice showed a significant intratumor amount of IL-18 compared

to mice treated with the vehicle (Fig.

4

c), whereas staining of

tumors from il18

−/−

mice revealed no staining (Supplementary

Fig. 9a). Concordantly, serum levels of IL-18 were statistically

more abundant in mice treated with HEI3090 than in vehicle

mice (Fig.

4

d), and no IL-18 was detected in the serum of mice

that received IL-18 neutralizing antibody. In addition, HEI3090

was unable to modulate the levels of IL-18 in p2rx7

−/−

mice.

Moreover, HEI3090-treated WT and p2rx7

−/−

mice show indeed

a significant difference in the release of IL-18. Finally, primary

peritoneal macrophages and bone-marrow-derived dendritic cells

(BMDC) from WT mice-cultured ex vivo with ATP and HEI3090

produce more IL-18 than cells cultured with ATP and vehicle

(Fig.

4

e and Supplementary Fig. 10c). IL-18 release by HEI3090

required the NLRP3 inflammasome, since its production is

inhibited by the NLRP3 inflammasome-specific inhibitor

(MCC950) (Fig.

4

e). Moreover, we showed that HEI3090

enhanced caspase-1 cleavage (Supplementary Fig. 8) meaning

that HEI3090 was able to increase IL-18 production by enhancing

the activation of the NLRP3 inflammasome. Activation of P2RX7

by HEI3090 in macrophages from p2rx7

−/−

mice failed to

increase IL-18 secretion (Supplementary Fig. 9a) and no staining

was observed in LLC tumors from HEI3090-treated p2rx7

−/−

mice (Supplementary Fig. 9b). In agreement with the observation

Fig. 2 HEI3090 inhibits tumor growth and combined with immunotherapy ameliorates mice survival. a Prophylactic administration. Average tumor area and weight of LLC allograft after daily treatment with HEI3090. Curves showed mean tumor area in mm2_{± SEM (vehiclen = 28, HEI3090 n = 32, two-way}

Anova test, left panel) and graph showed tumor weight the day of sacrifice. Data are presented by violin plots showing all points with hatched bar corresponding to median tumor weight (vehicle_{n = 28, HEI3090 n = 32. Two-tailed Mann–Whitney test, right panel). b Therapeutic administration.} Average tumor area and survival curves of LLC allograft. HEI3090 started when tumors reached 10–15 mm2_{of size. Curves showed mean tumor area in}

mm2_{± SEM (n = 12, two-way Anova test, left panel and Mantel Cox test, right panel). c Combo treatment. Average tumor area of LLC allograft after}

HEI3090 andαPD-1 treatment. Spaghetti plots and survival curves of animals are shown (n = 16, Mantel Cox test). d Schematic illustration of treatment given to LSL-KrasG12Dmice. Representative images showing lung tumor burden (Bar= 2 mm upper panel and 500 µm lower panel) with tumor histopathology (n = 4). Average tumor burden of LSL-KrasG12D mice in response to treatments were studied as the number of ADC per mouse (untreated, n = 4, vehicle + αPD-1, n = 4, HEI3090 + αPD-1, n = 6; two-tailed Mann–Whitney test) and the surface of ADC lesions per lung. Each point represents one lesion, all lesions are shown to illustrate the heterogeneity (untreated and vehicle_{+ αPD-1, n = 4 mice, HEI3090 + αPD-1, n = 6, Two-tailed}

Mann–Whitney test). p values: *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file. AD adenoma, ADC adenocarcinoma.

that HEI3090 retained its antitumor activity in mice treated with

IL-1β neutralizing antibody, HEI3090 did not modify IL-1β

protein levels in serum (Fig.

4

d) and did not modulate IL-1β

secretion in macrophages cultured ex vivo (Supplementary

Fig. 9c, d).

Increased production of IL-18 was also observed in the

LSL-Kras

G12D

_{lung tumor mouse model. Indeed, cells within lesions of}

mice treated with HEI3090 combined with

αPD-1 expressed more

IL-18 than mice treated with

αPD-1 alone (Fig.

4

f). IL-18 protein

levels in serum of mice that received the combo treatment were

also increased by sixfold (Fig.

4

f and Supplementary Fig. 9e). As

described with the LLC tumor model, HEI3090 did not impact

the levels of IL-1β in this in situ genetic tumor mouse model

(Supplementary Fig. 9f).

Collectively, these results demonstrate that the antitumor effect

of HEI3090 is highly dependent on P2RX7 expression and on its

capacity to induce the production of mature IL-18 in the presence

of eATP.

IL-18 is required to increase antitumor functions of NK and

CD4

+

T cells. To identify which immune cells were involved in

the HEI3090-induced antitumor response, we performed

antibody-specific cell depletion experiments. While NK and

CD4

+

T cells depletions prevented HEI3090 treatment from

inhibiting tumor growth (Fig.

5

a, b), CD8

+

T cells depletion had

no impact on HEI3090 treatment efficacy (Fig.

5

a). To further

study the effect of HEI3090 treatment on these subsets, we

assessed their cytokine production within the TME. Analyses of

tumor infiltrating immune cells first revealed that HEI3090

treatment significantly increased their capacity to produce IFN-γ

(Fig.

5

c). To precisely evaluate which cells in the TME produce

IFN-γ, we studied the TIL subpopulation and determined the

ratios of IFN-γ to IL-10 production in each subset (Fig.

5

d). NK

and CD4

+

T cells were more biased to produce IFN-γ than the

IL-10 immunosuppressive cytokine. CD8

+

T cells were relatively

less prone to modification in this cytokine ratio profile upon

HEI3090 treatment. In addition, twofold more NK cells from

mice treated with HEI3090 degranulate after ex vivo

restimula-tion with LLC compared to NK from control mice (Fig.

5

e),

confirming their activation state, while no effect was noticeable

on CD8

+

T cells. These phenotypic and functional analyses of

intratumor immune infiltration suggested furthermore that

treatment with HEI3090 stimulates CD4

+

T cells and NK cells’

activation in the TME. Importantly, IL-18 neutralization

abro-gated the increase of the IFN-γ/IL-10 ratio by CD4

+

_{T cells and}

NK cells (Fig.

5

f), suggesting that its production is a direct

con-sequence of IL-18 release and signaling. We showed that DC and

IL-18 were necessary for HEI3090’s activity (Figs.

3

b and

4

a, b).

In vitro stimulation of splenocytes treated with BzATP and

HEI3090 did not increase IFN-γ production by T cells and NK

cells indicating that its higher production in the tumor of treated

mice is rather an indirect consequence of the therapy

(Supplementary Fig. 10a). Concordantly, CD45

+

cells, CD8

+

T cells, and NK cells in the TME of p2rx7

−/−

mice supplemented

with WT DC showed an increase in the IFN-γ/IL-10 ratio in the

HEI3090-treated mice (Supplementary Fig. 10b). This result

indicates that WT DC were able to produce IL-18 after the

adoptive transfer, since antitumor effector cells were more prone

to produce IFN-γ than the IL-10 immunosuppressive cytokine.

Finally, we uncovered that HEI3090 treatment of LLC tumor

bearing mice in vivo increased the expression of MHC-I and

PD-L1 by 2.2-fold (Fig.

5

g). However, when LLC cells were treated

in vitro with HEI3090, neither MHC-I nor PD-L1 expression

were increased. By contrast, IFN-γ induced the expression of

these two proteins (Supplementary Fig. 10d). Taken together, our

results suggest that the in vivo increase of MHC-I and PD-L1

expression is a consequence of IFN-γ upregulation driven by

IL-18. Finally, using the LSL-Kras

G12D

_{tumor mouse model, we}

showed that tumor cells from mice that received both HEI3090

and

αPD-1 expressed more PD-L1 than tumor cells from mice

treated with

αPD-1 only (Fig.

5

h). Altogether, our results indicate

that HEI3090 increases IL-18 production allowing the

recruit-ment and activation of NK and CD4

+

T cells and the production

of IFN-γ. In turn, IFN-γ stimulates expression of MHC-I and

PD-L1 on cancer cells, leading to an increased-tumor

immunogeni-city and an increased sensitivity to anti-immune checkpoint

inhibitors.

Combined with

αPD-1 antibody, HEI3090 cures mice carrying

LLC tumors and allows memory immune response. Combined

with an

αPD-1 antibody, HEI3090 cured 80% of

LLC-tumor-bearing mice (Fig.

2

d). To determine whether cured mice

devel-oped an antitumor immune memory response, they were

rechallenged with LLC tumor cells 90 days after the

first

inocu-lation and were maintained without any therapy as illustrated in

Fig.

6

a. All long-term-recovered mice were protected from LLC

rechallenge, whereas all age-matched control mice developed

tumors (Fig.

6

b). The rechallenged mice were still alive 150 days

after the initial challenge (Fig.

6

c), sustaining the hypothesis that

combo treatment effectively promoted an efficient antitumor

memory immune response. Our results suggested that CD8

+

T cells are not directly involved in the primary antitumor effect of

HEI3090 (see Fig.

5

a). Nevertheless, it is well-characterized that

these cells play a pivotal role in the host’s ability to mount an

Fig. 3 Immune cells mediate the antitumor activity induced by HEI3090. a Average tumor area of LLC allograft in_{p2rx7-deficient mice (p2rx7}−/−) after daily treatment with HEI3090 or after adoptive transfer of WT splenocytes and daily treatment with HEI3090. Curves showed mean tumor area in mm2_±

SEM (vehiclen = 13, HEI3090 n = 16 mice, vehicle + WT spleno n = 11, HEI3090 + WT spleno: n = 12, two-way Anova test). b Average tumor area of LLC allograft in_{p2rx7-deficient mice (p2rx7}−/−) after adoptive transfer of WT DCs and daily treatment with HEI3090. Curves showed mean tumor area in mm2_{± SEM (n = 8, two-way Anova test). c Tumor weight of animals from the study shown in a and b. Data are presented by violin plots showing all points}

with hatched bar corresponding to median tumor weight (vehiclen = 8, HEI3090 n = 10 mice, vehicle + WT spleno n = 10, HEI3090 + WT spleno n = 12, vehicle+ WT DC n = 13, HEI3090 + WT DC n = 12 mice, two-tailed Mann–Whitney test). d Characterization of immune infiltrate at day 12. Percentage of CD45+analyzed byflow cytometry among living cells within TME. Data are presented by violin plots showing all points with hatched bar corresponding to median tumor weight (vehiclen = 7, HEI3090 n = 8, two-tailed Mann–Whitney test). e Representative picture of CD8+cells recruitment in LSL-KrasG12D mice over six mice studied (bar= 100 µm) and quantification. Data are presented by violin plots showing all points with hatched bar corresponding to median CD8+T cells (four tumors per mouse,_{n = 4 mice per group, two-tailed Mann–Whitney test). f Representative images of CD3 staining in LLC} tumors over six mice studied (bar= 100-µm upper panel and 50-µm lower panel) and quantification data are presented by violin plots showing all points with hatched bar corresponding to median CD8+T cells (n = 6 per group, two-tailed Mann–Whitney test). g Percentage of regulatory T cells determined by_{flow cytometry as FOXP3}+CD4+among CD3+within LLC tumors. Data are presented by violin plots showing all points with hatched bar corresponding to median FOXP3+cells of CD3 cells (n = 8 per group, two-tailed Mann–Whitney test). Gating strategy is presented in Supplementary Fig. 12. h Proportion of PMN-MDSC among CD45+within LLC tumors. Data are presented by violin plots showing all points with hatched bar corresponding to median PMN-MDSC cells among CD45+cells (_{n = 7, per group. Two-tailed Mann–Whitney test). Full gating strategy is presented in Supplementary Fig. 12. i Gating} strategy (left panel) and ratio of NK, CD4+, or CD8+T cells on PMN-MDSC within LLC tumors (right panel). Data are presented by violin plots showing all points with hatched bar corresponding to median of indicated cells (n = 8 per group, Two-tailed Mann–Whitney test). p values: *p < 0.05, **p < 0.01, ****_{p < 0.0001. Source data are provided as a Source Data file. Spleno Splenocytes, DC dendritic cells, PMN-MDSC poly morpho nuclear-myeloid-derived} suppressor cells.

Fig. 4 HEI3090-induced IL-18 production is required to inhibit tumor growth. a Average tumor area of LLC allograft in WT mice injected with IL-1β and IL-18 neutralizing antibodies and daily treatment with HEI3090. Curves showed mean tumor area in mm2_{± SEM (vehiclen = 28, HEI3090 n = 32,}

HEI3090+ IL-1β treated n = 6, HEI3090 + IL-18 n = 8. Two-way Anova test). b Average tumor area and tumor weight of LLC allograft in il-18-deficient mice (il-18−/−) and daily treatment with HEI3090. Curves showed mean tumor area in mm2_{± SEM (vehiclen = 9, HEI3090 n = 10. Two-way Anova test,}

left panel) and graph showed tumor weight the day of sacrifice. Data are presented by violin plots showing all points with hatched bar corresponding to median tumor weight (vehiclen = 9, HEI3090 n = 10. Two-tailed Mann–Whitney test, right panel). c Representative images of IL-18 staining in LLC tumors of six mice studied. Bar= 50 µm. d Production of IL-18 and IL-1β in serum of treated mice determined by ELISA. Data are presented by violin plots showing all points with plain bar corresponding to median cytokine concentration (IL-18 production: vehiclen = 7, HEI3090 n = 8, HEI3090 + αIL-18 n = 6, vehicle p2rx7−/−_{n = 5, HEI3090 p2rx7}−/−_{n = 8, (IL-1β production: vehicle n = 13, HEI3090 n = 12, HEI3090 + αIL-18 n = 10, vehicle p2rx7}−/−_{n = 10, HEI3090}

p2rx7−/−_{n = 12. Two-tailed Mann–Whitney test). e Ex vivo production of IL-18 in primary peritoneal macrophages. Data are presented by violin plots}

showing all points with hatched bar corresponding to median cytokine concentration (no treatmentn = 4, HEI3090 n = 4, ATP 1 mM n = 4, ATP 1 mM + HEI3090_{n = 4, ATP 3 mM n = 13, ATP 3 mM + HEI3090 n = 13, ATP 3 mM + MCC950 n = 3, ATP 3 mM + HEI3090 + MCC950 n = 3. Two-tailed} Mann–Whitney test). f Representative images of IL-18 staining in lung tumor lesions from LSL-KrasG12Dmice over four mice studied (bar= 100 µm) and production of IL-18 in serum of LSL-KrasG12Dmice. Data are presented by violin plots showing all points with hatched bar corresponding to median IL-18 concentration (vehicle_{+ αPD-1, n = 4, HEI3090 + αPD-1, n = 6. Two-tailed Mann–Whitney test). p values: *p < 0.05, **p < 0.01, ****p < 0.0001. Source} data are provided as a Source Datafile.

antitumoral adaptative immune response

20

_{. To evaluate the}

involvement of secondary memory CD8

+

T cells response in

these mice, we sorted CD8

+

cells from age-matched naïve mice or

5 months (day 150) surviving rechallenged mice (see Fig.

6

a) and

injected them to naïve mice prior to inoculation of LLC tumor

cell in a 1/1 ratio. No treatment was given to mice. In this

experimental condition, tumor growth was reduced by twofold in

mice that received CD8

+

T cells isolated from cured mice

(Fig.

6

d), indicating that the combo therapy promoted a

func-tional immune memory response that partly depends on CD8

+

T cells.

We next characterized the mice that were cured for a very long

period (300 days), as illustrated in Fig.

6

e. First, to discriminate

between dormancy and eradication of tumor cells, we depleted

CD8

+

T cells from 300-day-old cured mice and followed mice

welfare in the absence of treatment (Fig.

6

f). In this condition, no

tumor relapse was observed during the 40 days of the experiment

and the weight of the mice remained constant, revealing that the

combo treatment efficiently eliminated tumor cells. Second, since

circulating CD8

+

T cells are actively involved in the immune

memory response

20

and participated in the HEI3090-induced

antitumor response (see Fig.

6

d), we investigated their

involve-ment in the long-term memory immune response. To do so,

340-day-old cured or age-matched naïve mice were inoculated with

LLC tumor cells in the absence of CD8

+

T cells. Both naïve- and

cured-age-matched mice developed tumors (Figs.

6

g, h).

How-ever, the tumor growth was significantly reduced in cured mice

and three out of the six mice did not have tumors (Fig.

6

g, right

panel). Cured mice survival was also significantly increased in

comparison to naïve mice (Fig.

6

i). Collectively, these results

suggest that circulating CD8

+

T cells participate in the antitumor

immune response induced by HEI3090.

P2RX7 is positively correlated with high infiltration of

anti-tumor immune cells in NSCLC patients. Using the lung

ade-nocarcinomas (LUAD) TCGA dataset, we analyzed the effect of

P2RX7 expression levels on the recruitment of cytotoxic immune

cells. We clustered tumors of 80 patients with all stage (I-IV) of

lung adenocarcinoma according to P2RX7 expression and showed

that high levels of P2RX7 expression correlated with an increased

immune response in LUAD patients, characterized by a high

mRNA expression of CD274 (PD-L1), IL1B, IL18, a signature of

primed cytotoxic T cells (defined by CD8A, CD8B, IFN-G,

GZMA, GZMB, PRF1) (Fig.

7

a). Accordingly, Gene set

enrich-ment analysis (GSEA) demonstrated a positive correlation

between high P2RX7 expression and the well-characterized

established signatures of

“adaptive immune response,”

“T-cell-mediated immunity,” “cytokine production” (Fig.

7

b).

Further-more, high P2RX7 expression is correlated with high levels of

CD274 (PD-L1), independently of the stage of the disease

(Fig.

7

c). Consistently, a significant reduced overall survival is

observed for P2RX7 hi, CD274 hi, and P2RX7 hi

+ CD274 hi

LUAD patients (Fig.

7

d), suggesting that high expression levels of

P2RX7 is sufficient to bypass immune responses in the presence

of high levels of CD274. Such a situation is considered to benefit

from anti-checkpoint blockade and/or strategies aiming to

reac-tivate immune responses, e.g., with an activator of P2RX7.

Indeed, only few cancer patients achieve a response with

anti-immune checkpoint administered as single-agent and combined

therapies to enhance antitumor immunity and bring a clinical

benefit for patients are actively tested. We showed in this study

that the combination of HEI3090 and

αPD-1 is more efficient to

inhibit lung tumor growth than

αPD-1 alone (see Fig.

2

c).

Discussion

We demonstrated in this study that activation of the purinergic

P2RX7 receptor represents a promising strategy to control tumor

growth. We developed a positive modulator of P2RX7, called

HEI3090, that stimulates antitumor immunity. HEI3090 induces

production of IL-18 by P2RX7-expressing immune cells, by

mainly targeting DC. IL-18 drives IFN-γ production to increase

tumor immunogenicity and reinforces NK and CD4

+

T cells

immune responses and generates protective CD8

+

T cells

responses from recidivism. Noteworthy, therapeutic association

of HEI3090 with

αPD-1 antibody synergizes to cure mice in the

LLC syngeneic model of lung cancer and elicits an antitumor

immunity. We also observed that the combo treatment is more

efficient than αPD-1 alone to inhibit tumor growth in the

LSL-KRas

G12D

_{lung tumor genetic mouse model. Lung tumor}

regression correlates with an increased immune cell infiltration,

more secretion of IL-18 within the TME and higher expression of

PD-L1 by tumor cells. Furthermore, this mode of action was

confirmed using the B16-F10 melanoma tumor model

(Supple-mentary Fig. 6). Collectively these results demonstrate that the

antitumor activity of HEI3090 follows the same rules in all tumor

models tested and highlight the strength of HEI3090 to reactivate

antitumor immunity.

The design of P2RX7’s modulators was based on a ligand-based

approach allowing the generation of a pharmacophore model.

One hundred and twenty compounds were generated and were

tested for their ability to enhance P2RX7’s activities; five of them

were able to do so. HEI3090 was the most promising and effective

compound of the

five and was therefore chosen for our study.

Other natural or synthetic molecules have been described to

facilitate P2RX7 response to ATP

21–23

_{. P2RX4 is another member}

of the P2X family that is described to regulate P2RX7’s activities

in macrophages. Recently Kawano et al. have shown that a

positive modulator of P2RX4, the ginsenoside CK compound

24

_,

calibrates P2RX7-dependent cell death in macrophages

25

_.

Therefore, we checked whether HEI3090 modulates P2RX4’s

activities, which is not the case (Supplementary Fig. 11).

Until now, neither of these molecules has been tested in cancer

models. Moreover, attempt to facilitate P2RX7 activation in the

field of oncology has been limited by the finding that P2RX7

variants expressed by some tumor cells may sustain their

pro-liferation and metabolic activity

2

_{. To explore this question, we}

analyzed P2RX7’s functional features in ex vivo lung cancer

Fig. 5 HEI3090 triggers antitumor responses mediated by IL-18-induced NK and CD4+T cells. a Average tumor weight of LLC allograft in WT mice injected with depleting antibody and daily treated with HEI3090. Data are presented by violin plots showing all points with plain bar corresponding to median tumor weight (vehiclen = 28, HEI3090 n = 32, HEI3090 + αCD8 n = 8, HEI3090 + αCD4 n = 8, HEI3090 + αNK1.1 n = 8. Two-tailed Mann_{–Whitney test). b Spaghetti plots of LLC allograft in WT mice injected with depleting antibody and daily treated with HEI3090. vehicle n = 28,} HEI3090n = 32, HEI3090 + αCD4 n = 8, HEI3090 + αNK1.1 n = 8. c Average of IFN-γ+cells among CD45+cells in LLC tumors. Data are presented by violin plots showing all points with plain bar corresponding to median of IFN-γ+cells among CD45+cells (vehiclen = 5, HEI3090 n = 8. Two-tailed Mann–Whitney test). d Representative dot plots of IFN-γ and IL-10 staining on TILs (left panel) and ratios of IFN-γ on IL-10 in the same positive cells of each TILs (right panel). Data are presented by violin plots showing all points with plain bar corresponding to median of the cytokine ratio vehiclen = 5, HEI3090n = 8. Two-tailed Mann–Whitney test). e Ex vivo degranulation assay of splenocytes from LLC tumor bearing mice. CD107a+cells in NK, CD4+, and CD8+T cells are shown. Data are presented by violin plots showing all points with plain bar corresponding to median % of CD107a+cells (vehiclen = 10 vehicle and HEI3090_{n = 8. Two-tailed Mann–Whitney test). f Ratios of IFN-γ on IL-10 in the same positive cells of each TILs of IL-18 neutralized mice.} Data are presented by violin plots showing all points with plain bar corresponding to median of the cytokine ratio (vehiclen = 12, HEI3090 n = 6. Two-tailed Mann–Whitney test). g Flow cytometry analyses of MHC-I and PD-L1 expression on CD45−cells in LLC tumors. Data are presented by violin plots showing all points with plain bar corresponding to median of positive cells over CD45 cells (vehicle_{n = 8, HEI3090 n = 4. Two-tailed Mann–Whitney test).} h Representative images of PD-L1 staining in cancer lesion of LSL-KRasG12Dmice representative of six mice studied. (Bar= 100 µm) and quantification. Data are presented by violin plots showing all points with hatched bar corresponding to median of positive cells over total cells (vehiclen = 7, HEI3090 n = 8. Two-tailed Mann_{–Whitney test). p values: *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.}

samples

26

_{and showed that P2RX7 is functional in leukocytes}

whereas it is nonfunctional in tumor cells. Considering that

P2RX7 is a pro-apoptotic receptor, it makes sense that tumor cells

express a nonfunctional receptor. Whether this nonfunctional

receptor

corresponds

to

the

non-conformational

P2RX7

(nfP2RX7), described to be expressed by tumor cells

27

, remains to

be determined as well as the effect of HEI3090 on nfP2RX7.

Despite the

finding that P2RX7 expression by immune cells

restrains tumor growth

9,10

, the use of specific P2RX7 antagonists

has been promoted to treat cancers on the basis that inhibition of

tumor cell proliferation would be more efficient

28,29

_.

Consider-able effort has been made to engineer-specific P2RX7

antago-nists

30

_{and two of them (A74003 and AZ10606120) inhibited B16}

tumor growth in immunocompetent mice

10

_{. However, to our}

knowledge, these compounds have not been tested to treat cancer

and have failed in the

first clinical trials to treat inflammatory and

pain-related diseases

30

_{. In addition, in the preclinical mouse}

model, we were unable to inhibit LLC and B16-F10 tumor growth

when we tested the GSK1370319A compound, a

well-characterized P2RX7 antagonist

19

_{. In line with this}

_{finding, our}

present results suggest that facilitation of P2XR7 is associated

with efficient antitumor immunity in two different models of

transplantable tumor (expressing moderate or higher level of

P2RX7) as well as in the LSL-KRas

G12D

genetic lung cancer

mouse model. These results illustrate the view that P2RX7

acti-vation, rather than inhibition, represents a promising strategy in

cancer immunotherapy to unleash the immune responses,

nota-bly in conjunction with anti-checkpoint blockade. Therapeutic

antibody represents another promising

field of investigation to

treat cancer. In particular, Gilbert et al. described an antibody

against a nonfunctional P2RX7 variant that is promising to treat

basal cell carcinoma

31

_{. It would be interesting to combine}

HEI3090 with the therapeutic P2RX7 antibody and assay the

efficacy of this combo treatment.

Antineoplastic action of eATP was previously explored using

ATP administration in cancer patients and abandoned for lack of

convincing results

32,33

. Extracellular ATP is naturally degraded to

adenosine by ectoenzymes, and adenosine is an

immunosup-pressive molecule

34

_{. To inhibit the production of adenosine,}

blocking antibodies against CD39 and CD73 ectoenzymes were

produced and tested in mouse cancer models but also in ongoing

clinical trials (NCT03454451). This strategy seems to be

pro-mising, at least, in mice tumor models. In a

first study, Perrot

et al. showed that antibodies targeting human CD39 and CD73

promoted antitumor immunity by stimulating DC and

macro-phages which, in turn, restored the activation of effector T cells

35

_.

The authors also reported that the combination of anti-CD39

monoclonal antibody with oxaliplatin increased the survival of

tumor bearing mice, at least for 50 days. In a second study, an

independent anti-CD39 antibody was generated and tested on

different mouse tumor models. This antibody alone dampened

tumor growth and when combined with

αPD-1, it further slowed

tumor progression and 50% of the mice showed a complete

rejection

36

. Mechanistically, the anti-CD39 antibody treatment

led to an increased eATP levels via the P2RX7/NLRP3/IL-18 to

stimulate myeloid cells. Next, the authors demonstrated that

anti-CD39 antibody sensitized

αPD-1 resistant tumors by increasing

CD8

+

T cells infiltration. Our results confirm these findings but

also bring additional highlights. First, we showed that activation

of the eATP/P2RX7/NLRP3/IL-18 pathway by HEI3090 increased

long-lasting immune responses when combined with

αPD-1

antibody. Second, we demonstrated that the endogenous eATP

levels present in the TME were sufficient to enhance P2RX7’s

activation in the presence of HEI3090. These conditions are ideal

to allow P2RX7 activation where it is needed and avoid the

possible adverse effects associated with a systemic increase of

ATP levels, such as the one observed in response to anti-CD39

and -CD73 antibodies.

It was shown that eATP attracts DC precursors toward the

TME and promotes their activation state and their capacity to

present antigen

37,38

_{. During this study, we showed that HEI3090}

targets P2RX7-expressing immune cells, especially phagocytic

cells, such as macrophages and DCs (Supplementary Fig. 6a).

Between macrophages and DCs, DCs were the most promising

candidate; they express high levels of P2RX7, they are able to

release IL-18, and they are professional antigen-presenting cells

able to induce a potent antitumor immune response. We

there-fore tested their involvement by doing an adoptive transfer of WT

DC in p2rx7

−/−

mice. Doing so, we restored responsiveness to

HEI3090 (Fig.

3

b). We also observed that cDC CD4

+

from mice

treated with HEI3090 expressed higher levels of P2RX7

(Sup-plementary Fig. 4c). Collectively, these results demonstrated that

DCs mediate HEI3090’s antitumor activity, but macrophages may

have a secondary role in this effect.

Intriguingly, we did not observe an enhanced production of

mature IL-1β in mice treated with HEI3090 (Fig.

4

d). This was

unexpected as secretion of mature IL-1β depends on the ATP/

P2RX7-induced NLRP3 inflammasome activation as well

39

_.

However, unlike IL-1β, the inactive precursor form of IL-18 is

constitutively expressed in most human and animal cells.

Whe-ther this explanation is sufficient to account for this differential

IL-1β/IL-18 production is currently not known.

Whereas IL-1β is described to induce immune escape

40

_{, IL-18}

is involved in Th1 polarization and NK cell activation. We

showed here that IL-18 produced in response to HEI3090

treat-ment orchestrated the antitumor immune response by driving

IFN-γ production by NK and CD4

+

_{T cells. This is in line with}

the well-known IFN-γ stimulating activity of IL-18 (originally

designated as IFN-γ-inducing factor), and with its Th1 and NK

cells stimulating activity

41,42

. Protective effect of IL-18, but also

the activation of NLRP3, have been previously reported in various

mouse cancer models

43,44

_{NLRP3 activation in DCs as well as}

IL-18 have been linked to better prognosis, to drive antitumor

immunity and to enhance the efficacy of immunotherapies in

different tumor models

45,46

_{. In fact, when we combined HEI3090}

with an

αPD-1 antibody, we observed that the combo therapy

efficiently controlled tumor burden in the three cancer models

studied. Notably, the combo treatment cured 80% of LLC tumor

bearing mice and very interestingly, cured mice developed an

antitumor memory response.

CD8 memory T cells, comprising the circulating memory pool

—composed of effector memory (T

EM

) and central memory

(T

CM

) cells—and the tissue resident (T

RM

) pool, play crucial roles

in antitumor memory responses

47

_{. We showed that circulating}

CD8

+

T cells participated in cancer immunosurveillance after

HEI3090 treatment (Fig.

6

d). However, this CD8

+

T cells pool

cannot be responsible for the entire response, since antitumor

responses were still effective when CD8

+

T cells were depleted

(Fig.

6

g). These results suggest that other immune cells participate

in local cancer surveillance. Possible candidates are the

non-recirculating CD8

+

T

RM

cells. The persistence of T

RM

cells in

tissues has been shown to depend on signaling programs driven

by TGFβ and Notch-dependent signaling signature. Whether

HEI3090 directly stimulates those programs remains to be

Fig. 6 HEI3090 combined with_{αPD-1 induces antitumor memory immune response. a Schematic illustration of treatments with transfer of CD8 cells.} b Average tumor area of LLC allograft in 90-day-old WT and 90-day-old cured mice in absence of treatment. Curves showed mean tumor area in mm2_±

SEM (n = 7 per group, two-way Anova test). c Survival curves of animals from the study shown in b. Curves showed survival (n = 7 per group, Mantel Cox test).d Average tumor area of LLC allograft in WT mice injected with CD8+T cells isolated from rechallenged cured mice as shown ina. Curves showed mean tumor area in mm2_{± SEM (n = 4 per group, two-way Anova test). e Long-lasting antitumor immune response: schematic illustration of treatments.}

f Mouse body weight follow up of 300-day-old cured mice injected with anti-isotype (black circle) or depletingαCD8 antibodies (blue circle). Each curve represents one mouse (n = 6 per group). g Individual survival curves of 340-day-old WT and cured animals injected with anti-CD8 antibody and rechallenge with LLC in absence of treatment. (n = 6 per group). h Average tumor area from animals shown in g. Data are presented by violin plots showing all points with hatched bar corresponding to median of tumor area (n = 6 per group. Two-tailed Mann–Whitney test). i Survival curves from animals shown ing. (n = 6 per group, Mantel Cox test). p values: *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data_file.

determined but we observed, using HEK mP2RX7 cells, that

HEI3090 enhanced ATP-stimulated ERK pathways. Our results

are also compatible with a role for CD4

+

T memory cells and the

setup of a humoral response, in which B lymphocytes produce

antibody against tumor cells.

Therapy with different

αPD-1/PD-L1 antibodies was approved

in NSCLC in the

first- and second-line settings. However, a

significant fraction of patients does not benefit from the

treat-ment (primary resistance), and some responders relapse after a

period of response (acquired resistance)

48

. Expression of PD-L1

per se is not a robust biomarker with a predictive value since the

αPD-L1 response has also been observed in some patients with

PD-L1-negative tumors. Improvement of patient management for

immunotherapy undoubtedly relies on the identification of such

Fig. 7 P2RX7 expression in LUAD is associated with“hot” immunophenotype signature. a Association of P2RX7 mRNA expression with a cluster of inflammatory genes (heatmap). Expression values are represented as colors, where the range of colors (red, pink, light blue, dark blue) shows the range of expression values (high, moderate, low, lowest). Raw_{p values (Linear models for microarray analysis, Limma) are shown. b Gene set enrichment analysis} (GSEA) plot associatingP2RX7 high mRNA levels from LUAD patients (TCGA) with three inflammatory signatures. The enrichment score is shown as a green line, and the vertical black bars below the plot indicate the positions of specific inflammatory signature-associated genes, which are mostly grouped in the fraction of upregulated genes. For each signature, normalized enriched score (NES),_{p values (bilateral Kolmogorov–Smirnov), and false discovery} rate (FDR) are shown.c. Correlation curves ofP2RX7 and CD274 expression from LUAD patients (TCGA) of all stage (left panel), low stage (middle panel), and high stage (right panel).r, r2_{, andp values are shown in each panel, (Person correlation and t test). d Kaplan–Meyer plot (http://kmplot.com) showing}

survival curves of_{P2RX7 high vs. P2RX7 low patients (left panel), CD274 high vs. CD274 low (middle panel), and P2RX7 high or low vs. CD274 high or low} (right panel). For all panels, the optimal cutoff is determined on KMplot. Thep value (log-rank, Mantel Cox test), the hazard ratio, and number of patients are indicated. Source data are provided as a Source Datafile. ADC adenocarcinoma, HR hazard ratio.

predictive markers. Using TCGA dataset, we uncovered that

P2RX7 expression is correlated to CD274 (PD-L1) expression and

“hot” immunophenotype signatures in NSCLC patients. In

addition, patients with high P2RX7 and low CD274 or high

CD274 and low P2RX7 have a better overall survival than patients

with high CD274 and high P2RX7. This result suggests that

immunotherapies may be efficient in double positive patients and

questions the ability of P2RX7 to represent a valuable biomarker

for

αPD-1/PD-L1 therapies. In this context, we showed in another

study

26

_{that the expression of P2RX7B splice variant in tumor}

immune cells is associated with less infiltrated tumors in lung

adenocarcinoma. Mechanistically, we observed that the

differ-ential expression of the P2RX7B splice variant in immune cells

within tumor area correlates with the expression of a less

func-tional P2RX7 and lower leukocytes recruitment into LUAD.

We demonstrated that a small-molecule activator of P2RX7

boosts immune surveillance by unleashing the effector functions

of adaptive immune T cells and improving the efficacy of αPD-1

treatment. This therapeutic strategy holds new hopes for cancer

patients; by increasing tumor immunogenicity, it could

first

increase the number of patients eligible to immunotherapies and

second, it could also be used as a neoadjuvant or adjuvant

therapies of locally advanced lung tumors.

Methods

Mice. Mice were housed under standardized light–dark cycles in a temperature-controlled air-conditioned environment under specific pathogen-free conditions at IRCAN, Nice, France, with free access to food and water. All mouse studies were approved by the committee for Research and Ethics of the local authorities (CIEPAL #28, protocol numbers MESRI 23707, 13656) and followed the European directive 2010/63/UE, in agreement with the ARRIVE guidelines. Experiments were performed in accord with animal protection representative at IRCAN. P2rx7−/−(B6.129P2-P2rx7tm1Gab_{/J) and il18}−/−_{mice were from the Jackson} Laboratory. LSL-KRasG12D_{are from the Jackson Laboratories (ref 008179).} P2rx7-flox mice were engineered as follow: ES clones (C57/BL/6) containing a construct for the conditional elimination or re-expression of P2RX7 (purchased from The European Conditional Mouse Mutagenesis Program) were injected into blastocytes of C57Bl6/N, chimeric mice were selected and crossed with deleter mice that are transgenic for the Flip-recombinase under the control of the ubiquitous Actin promoter to produce p2rx7loxP/loxP_{mice. Our p2rx7}loxP/loxP_{mice, with loxP} sequencefloxing the second exon of p2rx7, were crossed with (C57BL/6NTacGt (ROSA)26Sor<tm1(ACTB-Cre,-EGFP)) transgenic mice which express the Cre recombinase under the control of the b-actin promoter to produce p2rx7exon2−/− mice or with LysM-Cre mice (B6.129P2-Lys2tm1(cre)lfo from the Jackson Laboratories (obtained from Dr B. Chazaud, France) to generate myeloid cell conditional p2rx7 knockout and WT control (p2rx7loxp/loxp_{) mice. Control C57BL/} 6J OlaHsD female (WT mouse) was supplied from Envigo (Gannat, France).

In vivo treatments. Five 105_{tumor cells were injected s.c. into the leftflank of WT} mice. Pharmacokinetic analysis (Fig.1g), to characterize the clearance of HEI3090 showed that after a period of 18-h HEI3090 concentration is <10 nM. Therefore, we have decided to inject HEI3090 daily. Mice were treated i.p. with vehicle (PBS, 10% DMSO) or with HEI3090 (1.5 mg/kg in PBS, 10% DMSO), which corresponds to the highest soluble dose. For therapeutic settings, treatment started at day 3, when tumor reached ~10–15 mm2_{, for a maximum of 20 days and} mice received vehicle or HEI3090 (3 mg/kg in PBS, 10% DMSO) daily. Depleting and neutralizing antibodies from BioXCell were given i.p. in the rightflank at days −1, 3, 7, and 10. αPD-1 antibody was given i.p. at days 4, 7, 10, and 13 (or as stated in the legend of thefigure) post-tumor cell inoculation. Antibodies are listed in Supplementary Table 1.αPD-1 and HEI3090 were injected separately, with at least 30 min delay between the two injections. Two hundred microliters liposome clo-dronate (Liposoma) were injected i.p. 3 days before LLC tumor cell inoculation in WT mice and then every 3 days, at least 1 h before HEI3090 treatment after the treatment started. CD8+T cells were sorted from peripheral lymph nodes of cured or naïve WT mice with Dynabeads®Untouched™ Mouse CD8 Cells (Invitrogen) according to the supplier’s instructions. 5.105_CD8+_{T cells were adoptively} transferred into 8-week-old naïve WT mice (i.v.) 1 day before tumor inoculation. 5.105_{LLC cells were injected s.c. into the leftflank of these mice and given no} further treatment.

Intratracheal delivery of adenoCre induces oncogenic KRAS in lung airway cells, leading to multifocal adenocarcinomas and a median survival of about 6 months49_{. Starting with tumors established for 3 months in adult LSL-Kras}G12D mice of either gender, treatment with vehicle or 1.5 mg/kg HEI3090 daily by i.p. injection was performed for 21 additional days.

Adoptive transfer inp2rx7-deficient mice. Spleens from WT C57BL/6J female mice were collected and digested in RPMI 1640 medium containing 5% FCS, 1-mg/ ml collagenase IV (Sigma-Aldrich), and 50 U/ml DNase I (Roche) for 7 min at 37 °C. Single-cell suspensions of spleens were prepared by passage through 100 µm cell strainers (BD Biosciences) and counted. For WT DCs isolation, spleens were digested with the spleen dissociation kit (Miltenyi Biotech) and isolated with the CD11c Microbeads UltraPure (Miltenyi biotech) according to the supplier’s instructions. 5.106_{splenocytes or 1.2.10}6_{DCs were injected i.v. in p2rx7}−/−_mice 1 day before subcutaneous injection of 5.105_{LLC cells into the leftflank. Mice were} treated i.p. every day for 12 days with vehicle (PBS, 10% DMSO) or with HEI3090 (1.5 mg/kg in PBS, 10% DMSO). At day 12, tumors were collected, weighted, and digested, whenflow cytometry analyses were done.

Flow cytometry and antibodies. Tumors were mechanically dissociated and digested with 1-mg mL−1collagenase A and 0.1-mg mL−1DNase I for 20 min at 37 °C. Then single-cell suspensions of tumors were prepared by passage through 100 µM cell strainers (BD Biosciences). Surface staining was performed by incu-bating cells on ice, for 20 min, with saturating concentrations of labeled Abs in PBS, 5% FCS, and 0.5% EDTA. After blocking Fc receptors using CD16/32 anti-bodies, cells were stained with the appropriate combination of antibodies (see Supplementary Table 1). The transcription factor staining Buffer Set (eBioscience) was used for the FoxP3 staining. For intracellular cytokines, staining was per-formed after stimulation of single-cell suspensions with Phorbol 12-myristate 13-acetate (PMA at 50 ng mL−1, Sigma), ionomycin (0.5μg mL−1_{, Sigma) and 1μL} mL−1Golgi Plug™ (BD Biosciences) for 4 h at 37 °C 5% CO2. Cells were incubated

with Live/Dead Near-IR stain (Invitrogen), according to the manufacturer’s pro-tocol prior to Ab surface staining. Then, intracellular staining was performed using Cytofix/Cytoperm™ kit (BD biosciences) following the manufacturer’s instructions. The production of IFN-γ and IL-10 was simultaneously analyzed in CD45+_{, NK,} CD4+T, or CD8+T cells. Datafiles were acquired and analyzed on Aria III using Diva software (BD Biosciences) or on the CytoFlex LX (Beckman Coulter) and analyzed using FlowJo software (LLC). Gating strategies are shown in Supple-mentary Fig. 12.

Immunohistological analysis of tumors. Collected tumors or lungs werefixed in 3% formamide for 16 h prior inclusion in paraffin. We used the following anti-bodies: anti-CD3, anti-CD8, anti-IL-18,αPD-L1, and anti-Ki67 (see Supplementary Table 1). After staining, slides were captured and analyzed using NDP view2-software. For the analyses,five zones per tumor were randomly selected and cells were counted using ImageJ software. Results are expressed as number of positive cells per total cell number.

Characterization of lung lesions in the LSL_-KRasG12Dmouse model. At the end of the treatment mice were sacrificed, exsanguinated and lungs processed for histologic and immunological analyses. After deparaffinization, HE stains were performed and slides were captured and analyzed using NDP view2 software. Tumor burden was calculated by determining the mean of total tumor area per lung using the NDP view2 software. To count the cells and determine the per-centage of Ki67-positive cells within lesions, ten lesions per lung, from grade 2 to 5 according to the Sutherland scoring50_{, were randomly selected, their perimeter was}

determined, and positive and negative nuclei were counted using ImageJ software. Results are expressed as number of cells per mm2_{and the percentage of} Ki67-positive cells.

Ex vivo macrophages and BMDC stimulation. Peritoneal lavage was done with RPMI 1640 medium on WT or p2rx7−/−mice. 4.105_{macrophages were seeded in a} 96-well plate overnight in RPMI 1640 containing 10% FBS, 2% sodium pyruvate, 1% penicillin/streptomycin, and 50-µMβ-mercaptoethanol. After two washes with the complete medium, cells were primed for 4 h with 100 ng/ml LPS (Sigma-Aldrich) at 37 °C and then stimulated for 30 min at 37 °C with ATP (Sigma-Aldrich) with or without 50 µM of HEI3090 or with 10 µM nigericin. When indicated, NLRP3 inflammasome was inhibited with 1 µM of MCC950 (Invivogen) for 1 h at 37 °C before cell stimulation. To prepare BMDC, leg bones were removed from C57Bl/6 mice, cut with scissors, andflushed with sterile PBS pH 7.4 via syringe. Bone-marrow-derived dendritic cells (BMDCs) were obtained from bone-marrow cells seeded in Petri dishes and cultured in RPMI medium containing 10% fetal calf serum, 2 mM L-Glutamine, 50-U/mL Penicillin, 50 µg/mL Streptomycin and 20% conditioned medium from GM-CSF-producing J558L cells. Medium was refreshed every 3 days. On the 7th day of the differentiation protocol, semi-adherent BMDCs were collected using PBS containing 10 mM EDTA and re-plated in new Petri dishes with fresh medium. Mature and semi-adherent BMDCs were used for experiments on the 14th day of culture. Supernatants were collected and stored at−80 °C before cytokine detection by ELISA using mouse IL-1 beta/IL-1F2 (R&D) and IL-18 (MBL) according to the supplier’s instructions.

Cells were lysed with Laemmli buffer (10% glycerol, 3% SDS, 10 mM Na2HPO4)

with protease inhibitor cocktail (Roche). Proteins were separated on a 12% SDS-PAGE gel and electro transferred onto PVDF membranes, which were blocked for 30 min at RT with 3% bovine serum albumin. Membranes were incubated with primary antibodies diluted 1/1000 at 4 °C overnight. The following antibodies were